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目的评价应用平行等位基因特异性序列检测技术(parallel allele-specific sequencing,PASS),对乙型肝炎病毒(HBV)进行耐药突变位点检测效果。方法应用PASS方法对17例未经抗病毒治疗和50例经抗病毒治疗失败的乙肝患者血浆进行耐药突变检测,其中50例乙肝抗病毒治疗失败的患者血浆同时采用测序法进行耐药分析。结果 17例未接受抗病毒治疗的患者样本中有8例检测到低比率的耐药突变位点(0.04%～16.89%),9例未发现有耐药突变。50例抗病毒治疗失败患者标本有7例PASS法和测序都未检测到耐药突变,16例PASS法检测到低比率耐药突变(0.03%～5.95%),测序法未检测到任何突变位点,27例PASS法和测序法都检测到了耐药突变(36%～100%)。应用PASS法进行连锁分析表明当多个耐药突变位点以高比率在同一患者体内检测到时,它们多以连锁的关系存在于同一病毒基因组。结论 PASS方法对检测HBV感染个体中耐药病毒群具有高度的灵敏性和特异性,并能对多个耐药突变位点进行连锁性分析。
Objective To evaluate the efficacy of parallel allele-specific sequencing (PASS) in detection of drug-resistant mutations in hepatitis B virus (HBV). Methods PASS was used to detect the mutation of 17 patients with hepatitis B without antiviral therapy and with 50 patients with failed hepatitis B virus antiviral therapy. 50 patients with failed hepatitis B virus therapy were analyzed by sequencing. Results Among the 17 patients who did not receive anti-virus therapy, 8 cases detected low rates of drug-resistant mutation sites (0.04% -16.89%), and 9 cases did not find any drug-resistant mutations. None of the 50 patients with failed antiviral therapy had drug resistance mutations detected by PASS and sequencing, 16 with low PASS (0.03% -5.95%), and no mutation Point, 27 cases of PASS and sequencing were detected drug-resistant mutations (36% to 100%). Linkage analysis using the PASS method showed that when multiple drug-resistant mutation sites were detected in the same patient at a high rate, they were mostly found in the same virus genome in a cascade relationship. Conclusion The PASS method is highly sensitive and specific for the detection of drug-resistant virus in HBV infected individuals, and can analyze the linkage of multiple drug-resistant mutation sites.